The alpha-ketoglutarate dehydrogenase complex (KGDHc), also known as the 2-oxoglutarate dehydrogenase complex, plays a crucial role in oxidative metabolism. It catalyzes a key step in the tricarboxylic acid (TCA) cycle, producing NADH (primarily for oxidative phosphorylation) and succinyl-CoA (for substrate-level phosphorylation, among others). Additionally, KGDHc is also capable of generating reactive oxygen species, which contribute to mitochondrial oxidative stress. Hence, the KGDHc and its dysfunction are implicated in various pathological conditions, including selected neurodegenerative diseases. The pathological roles of KGDHc in these diseases are generally still obscure. The aim of this study was to assess whether the mitochondrial malfunctions observed in the dihydrolipoamide succinyltransferase (DLST) and dihydrolipoamide dehydrogenase (DLD) double-heterozygous knockout (DLSTDLD, DKO) mice are associated with neuronal and/or metabolic abnormalities. In the DKO animals, the mitochondrial O consumption and ATP production rates both decreased in a substrate-specific manner. Reduced HO production was also observed, either due to Complex I inhibition with α-ketoglutarate or reverse electron transfer with succinate, which is significant in ischaemia-reperfusion injury. Middle-aged DKO mice exhibited minor cognitive decline, associated with microgliosis in the cerebral cortex and neuronal death in the Cornu Ammonis subfield 1 (CA1) of the hippocampus, indicating neuroinflammation. This was supported by increased levels of dynamin-related protein 1 (Drp1) and reduced levels of mitofusin 2 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in DKO mice. Observations on activity, food and oxygen consumption, and blood amino acid and acylcarnitine profiles revealed no significant differences. However, middle-aged DKO animals showed decreased performance in the treadmill fatigue-endurance test as compared to wild-type animals, accompanied by subtle resting cardiac impairment, but not skeletal muscle fibrosis. In conclusion, DKO animals compensate well the double-heterozygous knockout condition at the whole-body level with no major phenotypic changes under resting physiological conditions. However, under high energy demand, middle-aged DKO mice exhibited reduced performance, suggesting a decline in metabolic compensation. Additionally, microgliosis, neuronal death, decreased mitochondrial biogenesis, and altered mitochondrial dynamics were observed in DKO animals, resulting in minor cognitive decline. This is the first study to highlight the in vivo changes of this combined genetic modification. It demonstrates that unlike single knockout rodents, double knockout mice exhibit phenotypical alterations that worsen under stress situations.